Gemcitabine hcl

NSCLC cells, A549 and LNM35, were maintained in RPMI-1640 medium (Gibco, Paisley, UK) in a humidified incubator at 37 °C and 5% CO₂. The medium was supplemented with 1% of penicillin–streptomycin solution (Hyclone, Cramlington, UK) and 10% of fetal bovine serum (Hyclone, Cramlington, UK). Human umbilical vein endothelial cells (HUVECs) were maintained in an EndoGRO^TM-VEGF complete media kit (Merck Millipore, Massachusetts, USA) in a humidified incubator at 37 °C and 5% CO₂ in flasks coated with 0.2% gelatin. The culture medium of all cells was changed every 3 days, and cells were passed once a week when the culture reached 95% confluency for cancer cells and 80% for HUVECs.
Sodium DCA, cisplatin, camptothecin, gemcitabine HCl, erlotinib HCl and gefitinib were purchased from Sigma-Aldrich (Saint Louis, MO, USA). DCA was freshly dissolved in HyPure water (Hyclone, Cramlington, UK) before starting any experiment to make a stock solution of 1M, which was then diluted to the required concentrations for treatment.

Changes to drug target binding were quantified using a competitive binding assay between Gem-Atto and the parent drug, gemcitabine, on the established human pancreatic cancer cell lines: PANC-1, AsPC-1 and Capan-1. The cells were washed, trypsinized and plated in a 96-well glass bottom plate (Cellvis, Mountain View, CA) at a concentration of 0.1x10⁶ cells/ml. The cells equilibrated for 24 hours after which, gemcitabine-HCl (Sigma-Aldrich) was added to all cell lines at 0, 0.005, 0.05, 0.5, 5, 50 μM in triplicate wells. Every well also contained Gem-Atto at a single concentration (500 nM). Following a 24-hour incubation, the cells were gently washed 3 x 5 min with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde (PFA) for 15 min, washed again with PBS for 5 min and imaged in fresh PBS on a Zeiss AxioObserver inverted fluorescence microscope (Carl Zeiss, Oberkochen, Germany). For fluorescence excitation, a PhotoFluor II broad band light source (89 North, Burlington, VT) was filtered using a 650 ± 22.5 nm bandpass excitation filter and a 720 ± 30 nm bandpass emission filter to acquire images for Atto680. Individual cellular fluorescence was quantified using FIJI and analyzed with Prism (GraphPad, San Diego, CA).

ABC294640 (cGMP grade) was provided by Apogee Biotechnology Corporation. Gemcitabine HCl was purchased from Sigma-Aldrich. The following antibodies were purchased from: Cell Signaling - c-Myc (9402s), p21 (2947P), pRb (S780) (9307P), Rb (9309P), GAPDH (2118s), Santa Cruz - Ubiquitin (sc-8017) and Abcam - RRM2 (ab57653). The following primers were purchased from Qiagen: MYC (PPH00100B), RRM2 (PPH14649A), SPHK1 (PPH02491A), and SPHK2 (PPH21192A) GAPDH(PPH00150F).